Dipalmitoyl-sn-glycero-3-phosphocholine Research Articles

Effects induced by η6-p-cymene ruthenium(II) complexes on Langmuir monolayers mimicking cancer and healthy cell membranes do not correlate with their toxicity

The mechanism of chemotherapeutic action of Ru-based drugs involves plasma membrane disruption and valuable insights into this process may be gained using cell membrane models. The interactions of a series of cytotoxic η6-p-cymene ruthenium(II) complexes, [Ru(η6-p-cymene)P(3,5-C(CH3)3-C6H3)3Cl2] (1), [Ru(η6-p-cymene)P(3,5-CH3-C6H3)3Cl2] (2), [Ru(η6-p-cymene)P(4-CH3O-3,5-CH3-C6H2)3Cl2] (3), and [Ru(η6-p-cymene)P(4-CH3O-C6H4)3Cl2] (4), were examined using Langmuir monolayers as simplified healthy and cancerous outer leaflet plasma membrane models. The cancerous membrane (CM1 and CM2) models contained either 40 % 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 30 % cholesterol (Chol), 20 % 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), and 10 % 1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine (DPPS). Meanwhile, the healthy membrane (HM1 and HM2) models were composed of 60 % DPPC or DOPC, 30 % Chol and 10 % DPPE. The complexes affected surface pressure isotherms and decreased compressional moduli of cancerous and healthy membrane models, interacting with the monolayers headgroup and tails according to data from polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). However, the effects did not correlate with the toxicity of the complexes to cancerous and healthy cells. Multidimensional projection technique showed that the complex (1) induced significant changes in the CM1 and HM1 monolayers, though it had the lowest cytotoxicity against cancer cells and is not toxic to healthy cells. Moreover, the most toxic complexes (2) and (4) were those that least affected CM2 and HM2 monolayers. The findings here support that the ruthenium complexes interact with lipids and cholesterol in cell membrane models, and their cytotoxic activities involve a multifaceted mode of action beyond membrane disruption.

Sustained and improved enzymatic activity of trypsin immobilized in the Langmuir Blodgett film of DPPC: A rapid enzyme sensor for the detection of Azocasein

Immobilizing enzymes on biomimicking monolayer ultrathin films of phospholipids exhibit enhanced enzymatic activities (EAs) useful for biosensing applications. Preparation of such substrates with enzyme Trypsin (TRYP) immobilized in dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) matrix with enhanced and sustained enzymatic activity is a real challenge, as the isoelectric point of TRYP is reported to exist at pH ~ 10.5. This paper reports for the first time a facile procedure to fabricate smart substrates of TRYP entrapped in the DPPC monolayer using Langmuir–Blodgett (LB) technique. The incorporation of TRYP into DPPC monolayer at the air–water interface has been demonstrated from adsorption kinetics (π – t), room temperature surface pressure–area (π – A) isotherm plot and compressibility (β – π) studies. Circular dichroism (CD) and Grazing angle IR (GAIR) spectra signify changes in the secondary structure of incubated TRYP when immobilized in DPPC monolayer. Improved and sustained EA of TRYP upon its incubation on the organized monolayer LB films of DPPC has been observed. The reason behind such improvement in the EA of immobilized TRYP has also been suggested. The as fabricated substrates of immobilized TRYP can be used to sense soy milk, soy yoghurt, mayonnaise, meringue etc. using Azocasein as the protease substrate.

Visualization and measurement of the local absorption coefficients of single bilayer phospholipid membranes using scanning near-field optical microscopy.

Here we report the results of shear-mode thicknesses and absorption coefficient measurements made on neat membranes using scanning near-field optical microscopy (SNOM). Biomimic neat membranes composed of two different types of phoshpholipid molecules: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were found to exhibit different absorption coefficients under the SNOM. The localization of the lipids could be identified and correlated to the morphology of the membrane domains indicating that SNOM can be an effective and accurate approach for the label-free characterization of the structure-function relationships in cell membranes.

Constraining Polymers into β-Turns: Miscibility and Phase Segregation Effects in Lipid Monolayers.

Investigation of model biomembranes and their interactions with natural or synthetic macromolecules are of great interest to design membrane systems with specific properties such as drug-delivery. Here we study the behavior of amphiphilic β-turn mimetic polymer conjugates at the air–water interface and their interactions with lipid model membranes. For this endeavor we synthesized two different types of conjugates containing either hydrophobic polyisobutylene (PIB, Mn = 5000 g·mol−1) or helical poly(n-hexyl isocyanate) (PHIC, Mn = 4000 g·mol−1), both polymers being immiscible, whereas polyisobutylene as a hydrophobic polymer can incorporate into lipid membranes. The conjugates were investigated using Langmuir-film techniques coupled with epifluorescence microscopy and AFM (Atomic Force Microscopy), in addition to their phase behavior in mixed lipid/polymer membranes composed of DPPC (dipalmitoyl-sn-glycero-3-phosphocholine). It was found that the DPPC monolayers are strongly disturbed by the presence of the polymer conjugates and that domain formation of the polymer conjugates occurs at high surface pressures (π > 30 mN·m−1).

Effect of cell membrane fatty acid composition of Escherichia coli on the resistance to pulsed electric field (PEF) treatment

The underlying relationships between growth temperature, membrane fatty acid composition (mFAC) and PEF resistance of Escherichia coli (E. coli) were explored. Results indicated the PEF resistance of E. coli at stationary stage was directly related to mFAC. For the cells at stationary stage, as growth temperature decreased from 37 °C to 15 °C, the PEF (23.3 kV/cm, 1.2 ms) lethality of E. coli was increased 1.5 log10, reached 3.2 log10. Meanwhile, the proportion of unsaturated fatty acid (UFA) was increased from 20.07 to 47.65%. And the fluidity index (FI) of the cell membrane was also increased from 0.42 to 1.42. It revealed that the PEF resistance of E. coli at stationary stage was determined by the mFAC and fluidity of the cell membrane. Those conclusions were also confirmed by cytomembrane fluidity determined by fluorescence probe (DPH) and the different PEF-induced electro-permeabilization of dioleoyl-sn-glycero-3-phosphocholine (DOPC) and Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles. On the other hand, for the cell at exponential stage, the PEF resistance of E. coli was rarely affected by mFAC. The PEF (23.3 kV/cm, 1.2 ms) lethality of cells was almost the same (3.2 log10) at different growth temperature.

Effect of fatty acids on the permeability barrier of model and biological membranes

Because of the amphipathicity and conical molecular shape of fatty acids, they can efficiently incorporate into lipid membranes and disturb membrane integrity, chain packing, and lateral pressure profile. These phenomena affect both model membranes as well as biological membranes. We investigated the feasibility of exploiting fatty acids as permeability enhancers in drug delivery systems for enhancing drug release from liposomal carriers and drug uptake by target cells. Saturated fatty acids, with acyl chain length from C8 to C20, were tested using model drug delivery liposomes of 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and the breast cancer MCF-7 cell line as a model cell. A calcein release assay demonstrated reduction in the membrane permeability barrier of the DPPC liposomes, proportionally to the length of the fatty acid. Differential scanning calorimetry (DSC) and dynamic light scattering (DLS) experiments revealed that C12 to C20 fatty acids can stabilize DPPC liposomal bilayers and induce the formation of large structures, probably due to liposome aggregation and bilayer morphological changes. On the other hand, the short fatty acids C8 and C10 tend to destabilize the bilayers and only moderately cause the formation of large structures. The effect of fatty acids on DPPC liposomes was not completely transferrable to the MCF-7 cell line. Using cytotoxicity assays, the cells were found to be relatively insensitive to the fatty acids at apoptotic sub-millimolar concentrations. Increasing the fatty acid concentration to few millimolar substantially reduced the viability of the cells, most likely via the induction of necrosis and cell lysis. A bioluminescence living-cell-based luciferase assay showed that saturated fatty acids in sub-cytotoxic concentrations cannot reduce the permeability barrier of cell membranes. Our results confirm that the membrane perturbing effect of fatty acids on model membranes cannot simply be carried over to biological membranes of live cells.

The influence of NBD fluorescent probe on model membranes containing POPC and DPPC.

To investigate the effect of fluorescent probe on the properties of membranes, we studied model membranes composed of 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl 2-oleoyl-sn-glycero-3-phosphocholine (POPC) in the presence and absence of fluorescent probe. The morphology of giant unilamellar vesicles (GUVs) has been observed as a function of temperature and composition by fluorescence microscopy using NBD-DOPE or C6-NBD-PC as the probe. The phase behavior of model membranes containing no fluorescent probe was investigated by 2H-NMR spectroscopy. We found that the bright phase observed on GUVs was the fluid phase enriched in POPC and the dark phase was the gel phase enriched in DPPC. NBD-DOPE and C6-NBD-PC preferentially participated in the fluid-phase domains when GUVs were in the gel + fluid phase coexistence. Inclusion of both fluorescent probes (1 mol%) lowered the transition temperature of POPC/DPPC membranes. In addition, C6-NBD-PC exhibited a stronger effect than NBD-DOPE, which was considered to be associated with the structures of fluorescent molecules.

Effects of vesicle components on the electro-permeability of lipid bilayers of vesicles induced by pulsed electric fields (PEF) treatment

In this study, the effects of pulsed electric field (PEF) treatments on the electro-permeability of vesicles made from dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), dioleoyl-sn-glycero-3-phosphocholine (DOPC), Soya phosphatidylcholine (soya PC), cholesterol and a binary mixture of them encapsulated 5(6)-Carboxyfluorescein (5(6)-CF) were investigated, based on the electric field strength of 0–40 kV/cm and treatment time of 0–4.8 ms. Results indicated that the electro-permeability of lipid membrane was directly related to the composition of lipid bilayers membrane of vesicles. As compared to DPPC vesicles, DOPC vesicles were much more sensitive to PEF treatment. For the DOPC vesicles, the 5(6)-CF release percentage (R) was 9.24%, 20.12%, and 30.62% after PEF treatment at 20, 30, and 40 kV/cm for 4.8 ms treatment time, respectively. However, for the DPPC vesicles, it was only 1.84%, 4.02%, and 6.51%, respectively. On the other hand, the electro-permeability of DOPC vesicles was greatly affected by the addition of DPPC. The electro-permeability of DOPC vesicles was decreased sharply with the increase of DPPC molar ratio. Under the PEF treatment with 40 kV/cm and 1.2 ms, it was observed that the R of DOPC vesicles decreased markedly from 27.56% to 9.80% with the molar ratio of DPPC increased from 0 to 40%.For the soya PC vesicles, the R was dropped sharply from 48.13% to 24.11% with the molar ratio of cholesterol increased from 0 to 50%. Raman spectroscopy analysis revealed that the decrease of electro-permeability of soya PC vesicles with the addition of cholesterol was responsible for the incorporating of cholesterol into hydrocarbon partition of lipid bilayers, leading to the order arrangement of hydrocarbon chain of phospholipids, which was resulted an increase in the thickness of bilayers membrane and decrease in the fluidity of bilayers membrane. However, for the mixture of DOPC and DPPC (DOPC/DPPC) vesicles, there has no effect on the conformation of hydrocarbon chain of phospholipids and the fluidity of bilayer membrane of DOPC/DPPC vesicles with the addition of DPPC.

Strong Static Magnetic Fields Increase the Gel Signal in Partially Hydrated DPPC/DMPC Membranes.

It was recently reported that static magnetic fields increase lipid order in the hydrophobic membrane core of dehydrated native plant plasma membranes [Poinapen, Soft Matter 9:6804-6813, 2013]. As plasma membranes are multicomponent, highly complex structures, in order to elucidate the origin of this effect, we prepared model membranes consisting of a lipid species with low and high melting temperature. By controlling the temperature, bilayers coexisting of small gel and fluid domains were prepared as a basic model for the plasma membrane core. We studied molecular order in mixed lipid membranes made of dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) using neutron diffraction in the presence of strong static magnetic fields up to 3.5 T. The contribution of the hydrophobic membrane core was highlighted through deuterium labeling the lipid acyl chains. There was no observable effect on lipid organization in fluid or gel domains at high hydration of the membranes. However, lipid order was found to be enhanced at a reduced relative humidity of 43%: a magnetic field of 3.5 T led to an increase of the gel signal in the diffraction patterns of 5%. While all biological materials have weak diamagnetic properties, the corresponding energy is too small to compete against thermal disorder or viscous effects in the case of lipid molecules. We tentatively propose that the interaction between the fatty acid chains’ electric moment and the external magnetic field is driving the lipid tails in the hydrophobic membrane core into a better ordered state.

Phase Separation in Phosphatidylcholine Membrane Caused by the Presence of a Pyrimidine Analogue of Fluphenazine with High Anti-Multidrug-Resistance Activity

Phenothiazine compounds are known as effective inhibitors of a multidrug resistance (MDR) of tumor cells to chemotherapeutic agents. This group consists of many important substances used in human medicine such as antipsychotic drugs in the case of fluphenazine (FPh) or chlorpromazine (CPZ). Fluphenazine was on the World Health Organization (WHO) list of Essential Medicines of 2009, and its new pyrimidine analog (FPh-prm) presented in this work has been documented to have a high anti-MDR activity. In order to discover the character of alterations of the lipid bilayer structure caused by the presence of FPh-prm inside the lipid membrane, which is responsible for the essential increase of an anti-MDR activity of FPh-prm, microcalorimetric (differential scanning calorimetry), Laurdan fluorescence, (31)P nuclear magnetic resonance spectroscopy (NMR), and attenuated total reflectance Fourier transfer infrared spectroscopy (FTIR-ATR) were used for dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes mixed with a different concentration of amine analogue. It was stated that the formation of domains with different content of FPh-prm/DPPC can be a reason for the membrane-related mechanism of chemoprevention associated with the inhibition of the outward transport of anticancer drugs by the glycoprotein P (Pgp) in cancer cells by the pyrimidine analog of FPh. To our best knowledge, this report is the first to show the bilayer structure of domains formed by incomplete miscibility of fluphenazine-related compounds and phospholipid molecules. Our results provide a sound basis for the design of future modifications of anti-MDR drugs by providing very effective inhibitors of the pump activity of Pgp.

Highly Resolved Structure of a Floating Lipid Bilayer: Effects of Calcium Ions and Temperatue

Biological membranes are heterogeneous and highly dynamical organizations of lipids and proteins that define the outer boundary of a cell. Solid supported lipid bilayers have often been studied as model systems to understand the structures and properties of such cellular membranes. Such systems are constrained to a planar geometry and the strong support-membrane interaction could disrupt the inherent structural and functional properties of the membranes. To accurately mimic the biological membranes with their natural thermal fluctuations, curvature deformity and in-plane mobility of lipid molecules, a soft support is required. Hence, we have studied a double bilayer where the upper floating bilayer has the structural freedom to reproduce the morphology of a cellular membrane. Electron density profiles obtained from 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers by reflectivity studies using synchrotron x-rays provide unprecedented structural details of this system. Further, the effects of Ca2+ ions on the structure of these systems and the distribution of these ions near the bilayer have been investigated. These ions are preferentially bound to the head group region of the bilayer, which leads to a tight packing of lipids in the film. They cause the bilayer to thicken by increasing the hydrophobic core of the bilayer. Again, these ions are observed to intensify the flexibility of the bilayers, which is exhibited by the increased interfacial roughness. With the added Ca2+ ions, the inter bilayer separation is found to increase as a function of temperature and finally the floating bilayer unbinds from the adsorbed one. Such an effect was not observed in the absence of these ions.Acknowledgments. This work was supported by Office of Basic Energy Sciences, US Department of Energy, via Grant No. DE-FG02-04ER46173.

Zwitterionic lipid (DPPC)–protein (BSA) complexes at the air–water interface

Complexation of zwitterionic lipid, dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and protein, bovine serum albumin (BSA) at the air–water interface has been studied by surface pressure (π) – mean molecular area (A) isotherms and X-ray reflectivity. Although BSA has isoelectric point nearly at pH≈4.8, possibility of complex formation with lipid molecules has been investigated from low (≈4.0) to high (≈9.0) pH range in presence of divalent cation, Ca2+ in the water subphase. Both the isotherm and reflectivity analysis show that the interaction of BSA with lipid monolayer takes place from that low to high subphase pH range, i.e., complexation occurs both below and above of the isoelectric point. Only one layer of BSA forms below the lipid monolayer and the probable reasons for such complex formation have been proposed.

Enhanced gene transfection and serum stability of polyplexes by PDMAEMA-polysulfobetaine diblock copolymers

Polyethylene glycol or phosphorylcholine is often introduced into polycationic non-viral vectors to inhibit the non-specific protein adsorption. However the ability of vectors to condense DNA and the cellular internalization of complexes are unavoidably compromised. In this work, a polysulfobetaine-cationic methacrylate copolymer: 2-(dimethylamino) ethyl methacrylate-block-(N-(3-(methacryloylamino) propyl)-N,N-dimethyl-N-(3-sulfopropyl) ammonium hydroxide) (PDMAEMA-b-PMPDSAH) diblock copolymer was synthesized via atomic transfer radical polymerization method and investigated as a new non-viral vector for gene delivery. Incorporation of polysulfobetaine into cationic methacrylate retained a better DNA condensation capability. MTT assays revealed that the cytotoxicity of PDMAEMA 200-PMPDSAH n copolymer was lower than that of PDMAEMA 200. PDMAEMA 200-PMPDSAH 80 which was much superior to its homopolymer in mediating gene transfection demonstrated comparable efficiency to PEI25 kDa at a weight ratio of 8 in the presence of 10% serum. At higher serum contents, the transfection of PDMAEMA 200 and PEI25 kDa was deteriorated, whereas PDMAEMA 200-PMPDSAH 80 still retained better transfection efficiency, 4–5 fold more effective than PEI25 kDa. For the sake of comparative study, we synthesized structurally similar copolymer from DMAEMA and 2-methacryloyloxyethyl phosphorylcholine, PDMAEMA 200-PMPC 80. PDMAEMA 200-PMPDSAH 80 exhibited much higher gene transfer levels than PDMAEMA 200-PMPC 80 under the same conditions. The results of flow cytometry indicated that highly hydrophilic MPC block profoundly impeded the cellular internalization of nanocomplexes; in contrast, incorporation of polysulfobetaine remained the increased cellular uptake. Differential scanning calorimetry assay of thermodynamic phase transition of dipalmitoyl- sn-glycero-3-phosphocholine(DPPC) induced by polymer vectors demonstrated that MPC only marginally contributed to the perturbation of DPPC; polysulfobetaine facilitated more evident perturbation of DPPC bilayer instead, an indication that polysulfobetaine units could aid in the endocytosis of nanocomplexes.

Lipid–polyelectrolyte complexes at the air–water interface for different lipid packing

In presence of calcium ions in the water subphase, DNA and carboxymethyl cellulose (carboxyMC) can form complexes with the zwitterionic lipid dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) at the air–water interface. Depending upon the lipid packing, these polyelectrolytes can penetrate the lipid monolayer or can stay below the lipid monolayer after forming lipid–polyelectrolyte complexes, which are evidenced from the variation of the surface pressure with time at a constant area. Variation of surface pressure shows that at relatively less packing of DPPC molecules, (area per molecule is 119.5 Å 2 in presence of DNA and 101.1 Å 2 in presence of carboxyMC) surface pressure increases with time following the Langmuir growth relation. At intermediate packing, in presence of DNA, surface pressure decreases slowly compared to that of pure DPPC monolayer, whereas pressure remains nearly constant in presence of carboxyMC in the water subphase. At dense packing, i.e., at lower area per molecule (nearly 53 Å 2 in presence of both DNA and carboxyMC), surface pressure decreases slowly for both DNA and carboxyMC, compared to that of pure DPPC monolayer. Mostly all the decay curves fits well with the stretched exponential function or with the sum of one constant and two exponential decay terms, indicating the existence of different reorganization processes of the lipid and such lipid-polyelectrolyte complexes at the air-water interface.

Lipid Vesicle Aggregation Induced by Cooling

Lipid bilayer fusion is a complex process requiring several intermediate steps. Initially, the two bilayers are brought into close contact following removal of intervening water layers and overcoming electrostatic repulsions between opposing bilayer head groups. In this study we monitor by light scattering the reversible aggregation of phosphatidylcholine single shell vesicles during which adhesion occurs but stops prior to a fusion process. Light scattering measurements of dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in water show that lowering the temperature of about 0.14 micron single shell vesicles of DPPC (from 20 °C to 5 °C) and about 2 micron vesicles of DSPC (from 20 °C to 15 °C), but not of 1 micron vesicles of DMPC, results in extensive aggregation within 24 hours that is reversible by an increase in temperature. Aggregation of DSPC vesicles was confirmed by direct visual observation. Orientation of lipid head groups parallel to the plane of the bilayer and consequent reduction of the negative surface charge can account for the ability of DPPC and DSPC vesicles to aggregate. Retention of negatively charged phosphates on the surface and the burial of positively charged cholines within the bilayer offer an explanation for the failure of DMPC vesicles to aggregate. Lowering the temperature of 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS) vesicles from 20 °C to 5 °C failed to increase aggregation within 24 hours at Mg++/DPPS ratios that begin to initiate aggregation and fusion.

Early Stages of Oxidative Stress-Induced Membrane Permeabilization: A Neutron Reflectometry Study

Neutron reflectometry was used to probe in situ the structure of supported lipid bilayers at the solid-liquid interface during the early stages of UV-induced oxidative degradation. Single-component supported lipid bilayers composed of gel phase, dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and fluid phase, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), phospholipids were exposed to low-dose oxidative stress generated by UV light and their structures were examined by neutron reflectometry. An interrupted illumination mode, involving exposures in 15 min increments with 2 h intervals between subsequent exposures, and a continuous mode involving a single 60 (or 90) min exposure period were employed. In both cases, pronounced differences in the structure of the lipid bilayer after exposure were observed. Interrupted exposure led to a substantial decrease in membrane coverage but preserved its total thickness at reduced scattering length densities. These results indicate that the initial phase during UV-induced membrane degradation involves the formation of hydrophilic channels within the membrane. This is consistent with the loss of some lipid molecules we observe and attendant reorganization of residual lipids forming hemimicellar edges of the hydrophilic channels. In contrast, continuous illumination produced a graded interface of continuously varied scattering length density (and hence hydrocarbon density) extending 100-150 A into the liquid phase. Exposure of a DPPC bilayer to UV light in the presence of a reservoir of unfused vesicles showed low net membrane disintegration during oxidative stress, presumably because of surface back-filling from the bulk reservoir. Chemical evidence for membrane degradation was obtained by mass spectrometry and Fourier transform infrared spectroscopy. Further evidence for the formation of hydrophilic channels was furnished by fluorescence microscopy and imaging ellipsometry data.

Water confined by lipid bilayers: A multiplex CARS study

The orientational and hydrogen-bonding properties of water confined between lipid bilayers of a multilamellar vesicle of DPPC (dipalmitoyl- sn-glycero-3-phosphocholine) in D 2O have been investigated using multiplex coherent anti-Stokes Raman (CARS) microscopy. Spatially resolved analysis of the complete OD- and CH-stretch vibrational spectral region (2200–3200 cm −1) reveals that part of the (confined) interlamellar water has a weaker hydrogen-bond network than bulk water. Polarization dependent studies show negligible orientational order of the interstitial water molecules, in contrast to earlier CARS studies. Possible reasons for this discrepancy are explained in terms of interference between lipid and water resonances.

Interaction between O-carboxymethylchitosan and dipalmitoyl- sn-glycero-3-phosphocholine bilayer

O-carboxylmethylchitosan (OCMCS), a chitosan derivative, has emerged as a strong polymeric biomembrane perturbant. In this study, the interaction between OCMCS and dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) was examined with cross-polarization microscopy, differential scanning calorimetry (DSC) and the surface pressure–area isotherms techniques. Cross-polarized light images showed that OCMCS induced the fusion of small DPPC multilamellar vesicles (MLV) to form large lamellar structures. From DSC measurement, the highest degree of fusion was found at the optimum OCMCS concentrations between 0.0625 and 0.2 mg/ml which are orders of magnitude lower than those required for similar reductions with unmodified chitosan as perturbant. At these concentrations, the association of DPPC and OCMCS enhances the fusion of DPPC vesicles. Surface pressure–area isotherms of DPPC monolayer in the presence of OCMCS imply interactions between OCMCS and DPPC in neutral condition. In comparison with chitosan, OCMCS proved to be a more effective membrane perturbant not only in neutral but also in acidic and basic conditions. The physical driving forces for OCMCS-induced perturbation of DPPC bilayer in neutral conditions are mainly hydrogen bonding and hydrophobic interactions. In acidic or basic conditions, the physical driving forces are dominated by the electrostatic interactions. The strong OCMCS–DPPC interaction will potentially increase the effectiveness of OCMCS for gene or drug delivery.

N-alkylated chitosan as a potential nonviral vector for gene transfection.

Alkylated chitosans (ACSs) were prepared by modifying chitosan (CS) with alkyl bromide. The self-aggregation of ACSs in acetic acid solution was characterized by fluorescence spectroscopy and dynamic light scattering method. The results indicate that introducing alkyl side chains leads to the self-aggregation of ACSs, and CS with a 99% deacetylation degree shows no aggregation due to the electrostatic repulsion. The electrophoresis experiment demonstrates that the complex between CS and DNA was formed at a charge ratio (+/-) of 1/1; ACS/DNA complexes were formed at a lower charge ratio (+/-) of 1/4. A small amount of alkylated chitosans play the same shielding role as chitosan in protecting DNA from DNase hydrolysis. Differential scanning calorimetry (DSC) and atomic force microscopy (AFM) were employed separately to investigate the thermodynamic behavior of dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/CS and DPPC/ACS mixtures and the variation in topological structure of DPPC membrane induced by CS and ACS. It is shown that CS and ACS can cause the fusion of DPPC multilamellar vesicles as well as membrane destabilization. In contrast, the perturbation effect induced by ACS is more evident due to the hydrophobic interaction. CS and ACS were used to transfer plasmid-encoding CAT into C(2)C(12) cell lines. Upon elongating the alkyl side chain, the transfection efficiency is increased and levels off after the number of carbons in the side chain exceeds 8. It is proposed that the higher transfection efficiency of ACS is attributed to the increasing entry into cells facilitated by hydrophobic interactions and easier unpacking of DNA from ACS carriers due to the weakening of electrostatic attractions between DNA and ACS.

Interactions of phospholipid bilayer with chitosan: effect of molecular weight and pH.

Chitosan has demonstrated its potentials as a gene carrier and a membrane perturbant for subsequent drug delivery to cells. However, there is currently a lack of experimental correlation between the physiochemical properties of chitosan and the resulting degree of lipid bilayer destabilization. In this study, the effect of pH and chitosan molecular weight on the interaction between chitosan and dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayer was examined with cross-polarization microscopy, differential scanning calorimetry (DSC), and Fourier transform- (FT-) Raman spectroscopy. Cross-polarized images showed that the direct hydration of the DPPC/chitosan mixture led to the formation of larger DPPC multilamellar vesicles (MLV), and pure chitosan also induced fusions of individual MLV. Under the influence of chitosan, the calorimetric enthalpy of DPPC was reduced in a concentration-dependent manner, and a new phase appeared at 28 degrees C during sample cooling. Even the lowest chitosan mole fraction of 0.04% reduced the cooperative unit of the DPPC bilayer by more than 70%. In addition, the electrostatic effect between chitosan and DPPC tuned the degree of membrane bilayer perturbation. Reduction of pH increased the number of protonated amines on the chitosan backbone and caused further disruption on the membrane organization. Mixing DPPC with chitosan in an organic medium before hydration enhanced the hydrophobic interactions between the two molecules and greatly reduced the cooperative unit among individual lipids during the main phase transition. The increase of chitosan molecular weight also affected the cooperativity in the thermotropic transition of DPPC bilayer. FT-Raman spectroscopy provided additional evidence that chitosan directly perturbed the organizations of the hydrophobic inner core of the DPPC bilayer.